Chloroplast DNA assorts randomly in intraspecific somatic hybrids of Nicotiana debneyi

Summary Plants were regenerated following intraspecific fusion of leaf protoplasts from two naturally occurring genotypes of Nicotiana debneyi. The two genotypes differed in the EcoRl fragmentation pattern of chloroplast DNA and in the nuclear-coded phosphoglucomutase (Pgm) isozymes. There was no conscious selection for hybrid genotypes during protoplast culture or plant regeneration. Among 225 plants screened for Pgm, six were identified as nuclear hybrids. Restriction endonuclease and filter hybridisation analysis revealed that the cytoplasms of the hybrids contained one or other but never both parental chloroplast DNAs. The sorting out of chloroplasts was random and complete; the limit of detecting a rare chloroplast-DNA type in a mixture was 0.1%.     

Cytogenetic study of an interspecific cross of Nicotiana debneyi X N. umbratica

Summary Interspecific F₁ hybrids of Nicotiana debneyi Domin (2n=48) and N. umbratica Burbidge (2n=46), both belonging to the section Suaveolentes, showed a high degree of meiotic chromosome pairing. Two of the five F₂ plants obtained exhibited chromosome mosaicism. The first colchiploid generation (C₁) had the expected chromosome number of 2n=94 while C₂ showed 2n=88, a loss of three pairs of chromosomes. This same chromosome number continued in further colchiploid generations, followed up to C₅, except for a few plants in C₃ which showed chromosome mosaicism. The F₁ phenotype was stable through C₁ to C₅ and fertility was normal in colchiploids through all generations in spite of the loss of three pairs of chromosomes and chromosome mosaicism. This stability and fertility apparently reflect the tolerance of the genomes to the genetic adjustment of chromosome complements which is believed to be associated with the originally polyploid nature of the parental species and the chromosome doubling brought about in the amphidiploids.     

Nicotiana chloroplast genome

Summary RuBPCase, the enzyme responsible for carboxylation and oxidation of RuBP in a wide variety of photosynthetic organisms, is the major protein found in the chloroplast. Here we present the first evidence for direct expression in E. coli and B. subtilis of tobacco and Chlamydomonas ct-DNA sequences coding for the LS of RuBPCase as demonstrated by a simple in situ immunoassay.     

Nicotiana chloroplast genome

Summary A physical map containing six restriction sites of the Nicotiana tabacum chloroplast genome, together with the BamHI maps of N. tabacum, N. otophora and N. knightiana, and the SmaI maps of N. acuminata, N. plumbaginifolia, N. langsdorffii, N. otophora, N. tabacum, N. tomentosiformis and N. knightiana was constructed. In Nicotiana chloroplast genomes, the most frequently observed variations are point mutations. Deletions are also detected. Most of the observed changes are confined to one area of the large single copy region, which is designated as the hot spot. Based on the evidence obtained from Nicotiana chloroplast genomes, an origin of the inverted repeats in this genus is proposed. We suggest that the inverted repeats represent a vestige of what were once two identical, complete chloroplast genomes joined together in a head-to-head and tail-to-tail fashion, and that deletions generated the current chloroplast genome organization.     

Insertion polymorphism in pea chloroplast DNA

Summary The chloroplast DNA of higher plants is suitable for restriction endonuclease analysis due to its size and homogeneity. We have analysed 48 different cultivars of pea (Pisum sativum) with EcoRI and HindIII. Of these, only 24 show the standard genotype, the remaining 24 comprise four different classes of short insertions, three of which are found at the same site. Even though this kind of insertion polymorphism has not been detected elsewhere in the plant kingdom, it is consistent with the discovery that the chloroplast DNA of pea is destabilised through the loss of an inverted repeat.     

Extensive homologous chloroplast DNA recombination in the pt14 Nicotiana somatic hybrid

Summary In a previous study, six recombination sites have been confirmed in the chloroplast DNA (cpDNA) of pt14, a somatic hybrid of Nicotiana tabacum and Nicotiana plumbaginifolia. In the present study, physical mapping revealed six recombination sites in the 11.4-kb SalI fragment alone, only one of which has been previously identified. This fragment is located in the large unique region. We assume, therefore, that the pt14 cpDNA is a fine mosaic of the parental genomes with a recombination site about every 2 kb. A 748-bp region that comprised the intergenic region between ORF73 and ORF74B, and 460 bp of the petD intron have been sequenced. Parent-specific sequences in the pt14 DNA defined the regions within which recombination took place. The exact site of recombination events could not be determined because the parental sequences were identical between the polymorphic markers, and these sequences have been preserved in the pt14 line.     

Nicotiana chloroplast genome III. Chloroplast DNA evolution

Summary Nicotiana chloroplast genomes exhibit a high degree of diversity and a general similarity as revealed by restriction enzyme analysis. This property can be measured accurately by restriction enzymes which generate over 20 fragments. However, the restriction enzymes which generate a small number (about 10) of fragments are extremely useful not only in constructing the restriction maps but also in establishing the sequence of ct-DNA evolution. By using a single enzyme, Sma I, a elimination and sequential gain of its recognition sites during the course of ct-DNA evolution is clearly demonstrated. Thus, a sequence of ct-DNA evolution for many Nicotiana species is formulated. The observed changes are all clustered in one region to form a hot spot in the circular molecule of ct-DNA. The mechanisms involved for such alterations are mostly point mutations but inversion and deficiency are also indicated. Since there is a close correlation between the ct-DNA evolution and speciation in Nicotiana a high degree of cooperation and coordination betwen organellar and nuclear genomes is evident.     

Somatic hybrid plants of Nicotiana x sanderae (+) N. debneyi with fungal resistance to Peronospora tabacina

Background and Aims

The genus Nicotiana includes diploid and tetraploid species, with complementary ecological, agronomic and commercial characteristics. The species are of economic value for tobacco, as ornamentals, and for secondary plant-product biosynthesis. They show substantial differences in disease resistance because of their range of secondary products. In the last decade, sexual hybridization and transgenic technologies have tended to eclipse protoplast fusion for gene transfer. Somatic hybridization was exploited in the present investigation to generate a new hybrid combination involving two sexually incompatible tetraploid species. The somatic hybrid plants were characterized using molecular, molecular cytogenetic and phenotypic approaches.

Methods

Mesophyll protoplasts of the wild fungus-resistant species N. debneyi (2n = 4x = 48) were electrofused with those of the ornamental interspecific sexual hybrid N. × sanderae (2n = 2x = 18). From 1570 protoplast-derived cell colonies selected manually in five experiments, 580 tissues were sub-cultured to shoot regeneration medium. Regenerated plants were transferred to the glasshouse and screened for their morphology, chromosomal composition and disease resistance.

Key Results

Eighty-nine regenerated plants flowered; five were confirmed as somatic hybrids by their intermediate morphology compared with parental plants, cytological constitution and DNA-marker analysis. Somatic hybrid plants had chromosome complements of 60 or 62. Chromosomes were identified to parental genomes by genomic in situ hybridization and included all 18 chromosomes from N. × sanderae, and 42 or 44 chromosomes from N. debneyi. Four or six chromosomes of one ancestral genome of N. debneyi were eliminated during culture of electrofusion-treated protoplasts and plant regeneration. Both chloroplasts and mitochondria of the somatic hybrid plants were probably derived from N. debneyi. All somatic hybrid plants were fertile. In contrast to parental plants of N. × sanderae, the seed progeny of somatic hybrid plants were resistant to infection by Peronospora tabacina, a trait introgressed from the wild parent, N. debneyi.

Conclusions

Sexual incompatibility between N. × sanderae and N. debneyi was circumvented by somatic hybridization involving protoplast fusion. Asymmetrical nuclear hybridity was seen in the hybrids with loss of chromosomes, although importantly, somatic hybrids were fertile and stable. Expression of fungal resistance makes these somatic hybrids extremely valuable germplasm in future breeding programmes in ornamental tobacco.     

A point mutation in the chloroplast rps12 gene from Nicotiana plumbaginifolia confers streptomycin resistance

In an effort to understand the mechanism of streptomycin resistance in Nicotiana plumbaginifolia, we have sequenced the chloroplast rps12 gene, a potential molecular target. We report that a streptomycin-resistant mutant isolated from protoplast cultures of N. plumbaginifolia contains an A-to-G transition at nucleotide position 149 in exon 2 of the chloroplast rps12 gene. The detected point mutation predicts a substitution of arginine for lysine in a phylogenetically conserved region.     

A BamHI family of highly repeated DNA sequences of Nicotiana tabacum

Summary HRS60.1, a monomer unit (184 bp) of a highly repeated nuclear DNA sequence of Nicotiana tabacum, has been cloned and sequenced. Following BamHI digestion of tobacco DNA, Southern hybridization with HRS60.1 revealed a ladder of hybridization bands corresponding to multiples of the basic monomer unit. If the tobacco DNA was digested with restriction endonucleases which have no target site in HRS60.1, the larger part of DNA homologous to HRS60.1 remained as uncleaved relic DNA. These results suggest a tandem arrangement of this DNA repeat unit. Four other clones of tobacco nuclear DNA cross-hybridized with HRS60.1, thus forming a HRS60-family. Sequencing their inserts has shown their strong mutual homology. HRS60-family comprised about 2% of the nuclear genome of N. tabacum. Computer comparisons with other tandem plant-repeated DNA sequences could not detect any other homologous sequence.     

Inheritance and restriction fragment length polymorphism of chloroplast DNA in the genus Coffea L.

CpDNA variation among 52 tree samples belonging to 25 different taxa of Coffea and two species of Psilanthus was assessed by RFLP analysis on both the total chloroplast genome and the atpB-rbcL intergenic region. Twelve variable characters were distinguished allowing the identification of 12 different plastomes. The low sequence divergence observed might suggest that Coffea is a young genus. The results were in contradiction with the present classification into two genera. Additionally, cpDNA inheritance was studied in interspecific hybrids between C. arabica and C. canephora, and in an intraspecific progeny of C. canephora, using PCR-based markers. Both studies showed exclusively maternal inheritance of cpDNA.     

Physical maps of Nicotiana chloroplast DNA constructed by an efficient procedure

Summary The restriction profiles of chloroplast DNA (cpDNA) from Nicotiana tabacum, N. sylvestris, N. plumbaginifolia, and N. otophora were obtained with respect to AvaI, BamHI, BglI, HindIII, PstI, PvuII, SalI, and XhoI. An efficient mapping method for the construction of cpDNA physical maps in Nicotiana was established via a computer-aided analysis of the complete cpDNA sequence of N. tabacum for probe selection. The efficiency of this approach is demonstrated by the determination of cpDNA maps from N. sylvestris, N. plumbaginifolia, and N. otophora with respect to all of the above restriction endonucleases. The size and basic structure of the cpDNA from the three species are almost identical, with an addition of approximately 80 bp in N. plumbaginifolia. The restriction patterns and hence the physical maps between N. tabacum and N. sylvestris cpDNA are identical and there is no difference in the Pvull digests of cpDNA from all four species. Restriction site variations in cpDNA from different species probably result from point mutations, which create or eliminate a particular cutting site, and they were observed spanning the whole chloroplast molecule but highly concentrated in both ends of the large, single-copy region. The results presented here will be used for the forthcoming characterization of chloroplast genomes in the interspecies somatic hybrids of Nicotiana, and will be of great value in completing the exploration of the phylogenetic relationships within this already extensively studied genus.     

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Summary Each wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon-counting system     

The chloroplast genome of Nicotiana sylvestris and Nicotiana tomentosiformis: complete sequencing confirms that the Nicotiana sylvestris progenitor is the maternal genome donor of Nicotiana tabacum

The tobacco cultivar Nicotiana tabacum is a natural amphidiploid that is thought to be derived from ancestors of Nicotiana sylvestris and Nicotiana tomentosiformis. To compare these chloroplast genomes, DNA was prepared from isolated chloroplasts from green leaves of N. sylvestris and N. tomentosiformis, and subjected to whole-genome shotgun sequencing. The N. sylvestris chloroplast genome comprises of 155,941 bp and shows identical gene organization with that of N. tabacum, except one ORF. Detailed comparison revealed only seven different sites between N. tabacum and N. sylvestris; three in introns, two in spacer regions and two in coding regions. The chloroplast DNA of N. tomentosiformis is 155,745 bp long and possesses also identical gene organization with that of N. tabacum, except four ORFs and one pseudogene. However, 1,194 sites differ between these two species. Compared with N. tabacum, the nucleotide substitution in the inverted repeat was much lower than that in the single-copy region. The present work confirms that the chloroplast genome from N. tabacum was derived from an ancestor of N. sylvestris, and suggests that the rate of nucleotide substitution of the chloroplast genomes from N. tabacum and N. sylvestris is very low. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Inter- and intraspecific polymorphism at chloroplast SSR loci and the inheritance of plastids in Pinus radiata D. Don

DNA sequence analysis of chloroplast genomes has revealed many short nucleotide repeats analogous to nuclear microsatellites, or simple sequence repeats (SSRs). We designed PCR primers flanking five of these regions identified in the chloroplast sequence from Pinus thunbergii and tested them for amplification in Pinus radiata, P. elliotii, P. taeda, P. strobus, Pseudotsuga menziesii, Cupressus macrocarpa, four New Zealand native conifer species (Podocarpus totara, Podocarpus hallii, Podocarpus nivalis, Agathis australis), and four angiosperms (Vitex lucens, Nestegis cunninghamii, Actinidia chinensis, and Arabidopsis thaliana). A PCR product in the expected size range was amplified from all species and interspecific polymorphism was detected at all five loci. Intraspecific polymorphism was detected in P. radiata with four of the five primer pairs. One of these polymorphic chloroplast SSR (cpSSR) was then used to determine the inheritance of chloroplasts in 206 progeny from four control-pollinated, full-sibling P. radiata families. Approximately 99% of the progeny had the cpSSR variant of the pollen parent indicating that in Pinus radiata, like most other conifers, chloroplasts are typically inherited from the paternal parent. These results suggest that polymorphic chloroplast SSRs will be a valuable tool for studying chloroplast diversity, cyto-nuclear disequilibrium, and plastid inheritance in a range of species, and for the analysis of gene flow via pollen and paternity in species with paternal transmission of chloroplasts.     

Identification of two RAPD markers tightly linked with the Nicotiana debneyi gene for resistance to black root rot of tobacco

Linkage of randomly amplified polymorphic DNA (RAPD) markers with a single dominant gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris) of tobacco (Nicotiana tabacum L.), which was transferred from N. debneyi Domin, was investigated in this study. There were 2594 repeatable RAPD fragments generated by 441 primers on DNAs of Delgold tobacco, a BC₅F₈ near isogenic line (NIL) carrying the resistance gene in a Delgold background, and PB19, the donor parent of the resistance gene. Only 7 of these primers produced eight RAPD markers polymorphic between Delgold and PB19, indicating there are few RAPD polymorphisms between them despite relatively dissimilar pedigrees. Five of the eight RAPD markers were not polymorphic between Delgold and the NIL. All of these markers proved to be unlinked with the resistance gene in F₂ linkage tests. Of the remaining three RAPD markers polymorphic between Delgold and the NIL, two were shown to be strongly linked with the resistance gene; one in coupling and the other in repulsion. Application of the two RAPDs in the elimination of linkage drag associated with the N. debneyi resistance gene and marker-assisted selection for the breeding of new tobacco cultivars with the resistance gene is discussed.     

Nucleotide sequence of the chloroplast gene responsible for triazine resistance in canola

Summary The nucleotide sequence for the psbA gene from a triazine resistant cultivar of B. napus (cv Triton) has been determined. This gene encodes an open reading frame of 353 amino acids that is highly homologous to other higher plant psbA genes at both the nucleotide and amino acid levels. As has been found for other triazine resistant psbA genes, the Triton psbA contains an A to G nucleotide change which results in a serine to glycine amino acid substitution at position 264. The B. napus psbA gene also has a G insertion at position –9 resulting in a ribosome binding site sequence (AGGA) just before the initial methionine and suggesting that the entire open reading frame is translated. A large (72 bp) insertion is also found upstream of the B. napus psbA gene which resembles a similar insertion in the mustard psbA. The uncloneable nature of the entire gene is further investigated through reconstruction experiments and the implications discussed.     

Geographical distribution of two haplotypes of chloroplast DNA in four oak species (<Emphasis Type="Italic">Quercus</Emphasis>) in Japan

Chloroplast DNA polymorphism in four oak species (Quercus serrata, Q. mongolica var. crispula, Q. dentata and Q. aliena) was studied using collections from a total of 127 localities in Japan and South Korea on the basis of five intergenic spacers (trnD-trnT, trnT-trnL, rps14-psaB, trnS-trnT and trnQ-trnS). Although no variation existed in sequences among the four species, a single nucleotide (T/C) substitution in the trnQ-trnS intergenic spacer was found in all the four species, resulting in two haplotypes (T- and C-type). Phylogenetic analyses of the four species and related species showed that the C-type is derived and even likely of monophyletic origin, while the T-type is ancestral. Geographically, the T-type is widespread from South Korea to Japan, whereas the C-type is restricted to eastern Japan with rare exceptions. Eastern Japan approximately coincides with the distribution range of the boreal conifer forest during the last glacial maximum. Overall evidence suggests that the mutation from T- to C-type occurred in an individual of one of the four oak species and then was transferred to all the species by hybridization in eastern Japan, and that the Kanto District provided individuals with the C-type with a refugium during the last glacial maximum.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.